Method and Composition for Treating Heart Failure

ABSTRACT

A method and composition for treating, preventing or ameliorating heart failure, cardiac hypertrophy, and/or myocardial dysfunction includes administering a therapeutically effective amount of a HDAC inhibitor, such as phenylbutyrate, in combination with an ACE inhibitor, such as captopril.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation application of U.S. application Ser. No. 12/285,380, filed Oct. 3, 2008, which claims priority on prior U.S. Provisional Application Ser. No. 60/960,598, filed Oct. 5, 2007, both of which are hereby incorporated herein in their entirety by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The work leading to the present invention was supported by one or more grants from the U.S. Government, and specifically the VA merit review grant entitled “Restoration of Function in the Failing Heart”. The U.S. Government therefore has certain rights in the invention.

FIELD AND BACKGROUND OF THE INVENTION

The present invention is generally directed to cardiac therapy, and more particularly to treating, preventing, or ameliorating heart failure and/or cardiac hypertrophy, and/or dysfunction by reactivating silenced adult cardiac gene expression.

Heart failure is a leading case of death and disability. Hospitalization and long-term care for this condition represent major health care costs items. Among other entities, hypertension is a major factor underlying the development of heart failure.

Neither the cause of hypertension or mechanisms underlying heart failure are fully understood. It is clear, however, that increased neurohormonal activity accompanies heart failure and ameliorating this activity by beta adrenergic antagonists and inhibitors of the rennin-angiotensin aldosterone system improves clinical state. These treatments represent cornerstones in the management of heart failure today.

Although symptomatic improvement in heart failure patients is found with neurohumoral blockade, reversal of the underlying pathophysiology does not take place. Pathological events include adverse remodeling of the myocardium, associated with a modification of gene expression, an increase in left ventricular mass, depression of intrinsic myocardial function.

Conventional treatments for heart failure are designed to stabilize disease progression and are primarily limited to the administration of an angiotensin converting enzyme (ACE) inhibitor, angiotensin receptor blocker, beta adrenergic blocker, or diuretic. For example, a ACE inhibitor, such as captopril, is frequently administered to patients with hypertension and acutely decompensated heart failure. The efficacy of ACE inhibitors, such as captopril, is based on their ability to reduce circulation levels of angiotensin II, to thereby reduce mean arterial pressure and systemic vascular resistance. This results in decreased workload on the heart in patients with heart failure. This treatment may temporarily reduce clinical symptoms of heart failure, however, does not effectively treat the underlying disease and the long-term outlook for heart failure patients remains poor.

Much of the pathophysiology associated with heart failure may be due, in large part, to abnormal gene transcription that results from aberrant silencing of adult cardiac gene expression and recapitulation of the fetal gene program. Histone acetylase and deacetylaces can play a role in the control of gene expression. A prior patent document (US Patent Application Publication No. 2006/0025333) entitled “Inhibition of histone deacetylases as a treatment for cardiac hypertrophy” provides methods for treatment and prevention of cardiac hypertrophy in patients at risk of developing heart failure by administration of class II histone deacetylases (HDAC) inhibitors, consisting of tricoststin A, trapoxin B, MS 275-27, m-carboxycinnamic acid bis-hydroxamide, depudecin, oxamflatin, apicidin, suberoylanilide hydroxamic acid, Scriptaid, pyroxamide, 2-amino-8oxo-9,10-epoxy-decanoyl, 3-(4-aroyl-1H-pyrrol-2-yl)-N-hydroxy-2-propenamide and FR901228. However, it is noted that this patent document does not include phenylbutyrate. Aside from the actions of HDACs, when DNA is methylated in the promotor region of genes, where transcription is initiated, normal adult genes are inactivated and silenced (Reference 1), which can lead to recapitulation of the fetal gene profile that characterizes the failing heart.

As noted above, angiotensin converting enzyme inhibitors, the primary standard treatment for hypertension and heart failure, are designed to stabilize disease progression. However, myocardial dysfunction involving depressed myocardial contractility and cardiac enlargement due, in large part, to pathologic gene expression remains unresolved. An approach to selectively target cardiac gene transcription to alter gene expression, and thereby restore the adult cardiac profile, reduce ventricular mass and increase contractile function is needed to effectively treat heart failure.

Histone deacetylase inhibitors are substances causing inhibition of the activity of histone deacetylase, resulting in hyperacetylation and leading to chromatin relaxation and wide scale changes of gene expression. Current compounds shown to inhibit the activity of histone deacetylases fall into six structurally diverse classes. Phenylbutyrate (MW 164.21), comprises the short chain fatty acid class and is well-tolerated clinically at drug concentrations, which effect acetylation of histones in vitro. Phenylbutyrate has been used for the treatment of urea cycle disorders in children, sickle cell disease, thalassemia, cancer and more recently for the treatment of cystic fibrosis and ALS disease. However, thus far, phenylbutyrate has not been used for the treatment of heart failure.

OBJECTS AND SUMMARY OF THE INVENTION

The principal object of the present invention is to provide a method and composition for treating a patient and other mammals having heart failure.

Another object of the present invention is to provide a method and composition for reversing the underlying disease process for heart failure or cardiac hypertrophy.

Another object of the present invention is to provide a pharmaceutical composition and method for the treatment of acute and chronic heart failure.

Another object of the present invention is to provide a pharmaceutical composition that targets gene changes of the heart, and method for the treatment of myocardial dysfunction involving reestablishment of normal cardiac gene transcription to induce adult proteins and regress ventricular enlargement due to heart failure.

Another object of the present invention is to provide compositions and methods to treat patients and animals with congestive heart failure by administration of an ACE inhibitor and an effective amount of a combined inhibitor of histone deacetylase and DNA methylation.

Another object of present invention is a combined use of phenylbutyrate, a histone deacetylase inhibitor, together with a angiotensin converting enzyme inhibitor, such as captopril for the treatment of heart failure.

Another object of the present invention is to provide a new treatment for heart failure which accompanies the use of an angiotensin converting enzyme inhibitor, such as captopril (a standard treatment for the management of heart failure) with phenylbutyrate. While use of angiotensin converting enzyme inhibitors alone has been found to improve clinical state, the gene expression changes associated with heart failure are minimally modified. Left ventricular hypertrophy and myocardial contractile state are not improved. Adding phenylbutyrate to angiotensin converting enzyme inhibitor results in reversal of numerous gene expression changes, amelioration of left ventricular hypertrophy and contractile dysfunction. Thus, simultaneous administration of an angiotensin converting enzyme inhibitor to stabilize hemodynamics together with phenylbutyrate to modulate gene expression is demonstrated in the present invention to provide an effective method for the treatment of heart failure.

In summary, the invention employs a therapeutic strategy that utilizes a combination of drugs to modulate the expression of cardiac genes involved in the pathogenesis of heart failure. More specifically, compositions and methods are provided for treating aberrant silencing of adult cardiac gene expression and deactivation of the fetal gene profile associated with heart failure through inhibition of DNA methylation and histone deacetylation in combination with a sulfhydryl-containing angiotensin converting enzyme inhibitor. The method comprises administering to a patient or mammal suffering from heart failure a therapeutically effective amount of sodium phenylbutyrate, an inhibitor of histone deacetylase, which is metabolized in the human body by beta-oxidation to phenylacetate, a hypomethylating agent, in combination with an effective amount of the angiotensin converting enzyme inhibitor captopril. The present invention, therefore, provides method and composition for treating, preventing or ameliorating heart-failure and cardiac hypertrophy and dysfunction by reactivating silenced adult cardiac gene expression.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be more fully understood and further advantages will become apparent when reference is made to the following description of the invention and the accompanying Figures, in which:

The drawings are the result of data obtained from 6 animals per group. Age matched groups are 1) non-failing SHR, 2) SHR with heart failure, 3) failing SHR treated with captopril (2 g/L in the drinking water) for 30 days, and 4) failing SHR treated with phenylbutyrate (6 g/L in the drinking water) and captopril for 30 days.

FIGS. 1A-1B illustrate serial echocardiographic measurements in SHR. LV ejection fraction (FIG. 1A) and end-systolic and end-diastolic LV volume (FIG. 1B) of male SHR during the transition to heart failure (12, 17, and 19 months of age). EF, LV ejection fraction (%); ESV, end-systolic volume (ml); EDV, end-diastolic volume (ml). LV function remains compensated until approximately 17 months, and then rapidly declines; mean age at onset of heart failure 19±1 months.

FIGS. 2A-2B represent serial echocardiographic measurements (n=6 animals per group) of LV ejection fraction (LVEF) from individual SHR obtained before the onset of heart failure (Baseline), at the time of heart failure (HF) and following 30 days of treatment (Rx) with either captopril (CAPT—FIG. 2A) or combined captopril and phenylbutyrate treatment (CAPT+PB—FIG. 2B). Note marked reduction in LVEF with HF. There was marked improvement with combined treatment, as compared to minimal improvement with captopril alone.

FIG. 3 illustrates LV ejection fraction (LVEF) in non-failing SHR (SHR-NF), SHR with HF (SHR-F) and SHR-F following 30 days of captopril (CAPT) or combined phenylbutyrate and captopril treatment (CAPT+PB). There was no significant change in LVEF with CAPT. In contrast, LVEF with CAPT+PB increased to near control. Data are mean±SD (n=6 per group).

FIG. 4A illustrates LV/BW ratio, an index of LV hypertrophy, in non-failing SHR (SHR-NF), SHR with HF (SHR-F) and SHR-F following 30 days of captopril (CAPT) or combined treatment (CAPT+PB). There was no significant reduction in LV/BW with CAPT. In contrast, LV/BW with CAPT+PB was less as compared to untreated SHR, with or without HF. Data are mean±SD (n=6 per group).

FIG. 4B illustrates RV/BW ratio, an index of RV hypertrophy, in non-failing SHR (SHR-NF), SHR with HF (SHR-F) and SHR-F following 30 days of captopril (CAPT) or combined treatment (CAPT+PB). RV/BW was increased with HF, and decreased with treatment. Data are mean±SD (n=6 per group).

FIG. 5A illustrates identified transcripts whose expression was increased with HF and returned toward non-failing levels with treatment. Transcripts are grouped by functional categories based on Affymetrix gene ontology and biological process annotations. Solid bars: SHR-F, hatched bars: CAPT; stippled bars: CAPT+PB.

FIG. 5B illustrates identified transcripts whose expression was decreased with HF and returned toward non-failing levels with treatment. Transcripts are grouped by functional categories based on Affymetrix gene ontology and biological process annotations. Solid bars: SHR-F, hatched bars: CAPT; stippled bars: CAPT+PB.

FIG. 6A illustrates quick release force-velocity (F-V) relationships from LV papillary muscles. F-V relationships were significantly depressed (p<0.01) in SHR-F in comparison to SHR-NF. F-V relationships were not significantly increased with CAPT, but were significantly greater with CPT+PB. Data are mean±SD (n=6 per group).

FIG. 6B illustrates active tension (AT) of LV papillary muscles. AT Active tension was significantly depressed (p<0.01) in SHR-F in comparison to SHR-NF. CAPT did not improve AT compared to SHR-F. AT was significantly greater with CAPT+PB as compared to SHR-F and CAPT. Data are mean±SD (n=6 per group).

FIG. 7A illustrates failure effect. Total number of transcripts significantly different (p<0.05) between LV samples from SHR-F compared to SHR-NF.

FIG. 7B illustrates treatment effect. Total number of transcripts significantly different (p<0.01) between SHR-F and SHR-F with CAPT or CAPT+PB.

FIG. 8A illustrates failure effect. Total number of transcripts significantly different (p<0.05) between LV samples from SHR-F compared to SHR-NF.

FIG. 8B illustrates treatment effect. Total number of transcripts significantly different (p<0.01) between SHR-F and SHR-F with CAPT or CAPT+PB.

FIG. 9 illustrates the proposed mechanism by which phenylbutyrate is likely to modulate cardiac gene transcription.

FIG. 10 illustrates the primary pharmacological drug actions of captopril and phenylbutyrate.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT(S) OF THE INVENTION

The present invention discloses a composition and method for reestablishing adult cardiac gene transcription through inhibition of histone deacetylases and DNA methylation by the use of phenylbutyrate in combination with a angiotensin converting enzyme inhibitor, such as captopril or a pharmaceutically acceptable salt thereof, for the preparation of a pharmaceutical composition for the treatment of heart failure.

Based on studies in a genetic model of hypertension and heart failure the spontaneously hypertensive rat, a new treatment for heart failure is described, which reverses: 1) changes in cardiac gene expression associated with heart failure, 2) biventricular hypertrophy, and 3) myocardial dysfunction. The mechanism of treatment effect of phenylbutyrate, as an adjunct treatment, with standard angiotensin converting enzyme (ACE) inhibition (e.g. captopril) is suggested to be through its action as a histone deacetylase inhibitor. Phenylbutyrate is a non-specific histone deacetylase inhibitor that is already approved by the Food and Drug Administration to treat ornithine transcarbamylase deficiency.

Phenylbutyrate and its derivatives thereof are disclosed to be useful as adjunct agents for treatment in heart failure. Pharmaceutical formulations and the use of compounds of phenylbutyrate in combination with other agents (e.g. captopril) are also disclosed.

Phenylbutyrate is a natural nontoxic colorless tasteless aromatic fatty acid purified from mammalian urine and plasma, is Food and Drug Administration approved for children with hyperammonemia associated with inborn errors of urea synthesis.

In the course of our experiments, we discovered phenylbutyrate strongly affected cardiac gene expression, cardiac hypertrophy and contractile function of myocardial muscle with heart failure.

Phenylbutyrate is one of six structurally diverse classes of compounds that inhibit the activity of histone deacetylases (HDAC). Phenylbutyrate is of the short chain fatty acid class. Sodium phenylbutyrate is an FDA approved orphan drug (i.e. Buphenyl) for clinical use in urea cycle disorders such as ornithine transcarbamylase deficiency (Reference 2). In humans phenylbutyrate is metabolized by beta-oxidation to phenylacetate which is conjugated with glutamine to phenylacetylglutamine, that is eliminated with the urine. However, phenylacetate also exhibits hypomethylation activity (Reference 3) independent of its effects on nitrogen metabolism. In animals without heart failure oral administration of phenylbutyrate has been shown to inhibit histone deacetylase activity (References 4 and 5) and DNA methylation (Reference 6) at pharmacologic concentrations that results in numerous changes in tissue gene expression in vivo.

Captopril is a sulfhydral-containing analog of proline that competitively inhibits angiotensin converting enzyme, thereby decreasing circulating and tissue levels of angiotensin II, increasing plasma renin activity and decreasing aldosterone secretion to reduce blood pressure. Captopril is FDA approved as an antihypertensive drug for the treatment of hypertension.

A compound including a phenylbutyrate structure and a pharmaceutically acceptable angiotensin converting enzyme inhibitor (i.e. captopril) are mixed together as a single preparation for oral administration to simultaneously stabilize disease progression and target transcriptional changes of the heart in the treatment of humans and animals with heart failure.

The histone deactylase inhibitor, phenylbutyrate, can be used in the form of a pharmaceutically acceptable salt. As such it may be used so long as it does not adversely affect the desired pharmacological effects of the compound. Examples of pharmaceutically acceptable salts include, alkali metal salts such as sodium salt or a potassium salt, alkaline earth metal salts such as calcium salt or magnesium salt, salts with an organic base such as an ammonium salt, or a salt with no organic base such as triethylamine salt or an ethanolamine salt. Sodium-free glycerol-mono-phenylbutyrate ester could also be used in patients who might be harmed by large sodium load.

The use of a histone deacetylase inhibitor and angiotensin converting enzyme inhibitor or combined agent of the present invention may be administered in the form of soft and hard capsules, tablets, granules, powders, solutions, suspensions or the like. In the case of non-oral administration, they may be administered in the form of injections solution, drip infusion formulations or patches or the like, whereby continued membrane absorption can be maintained in the form of solid, viscous liquid, or suspension. The selection of the method for the delivery of these formulations and the vehicles, disintegrators or suspending agents, can be readily made by those skilled in the art. The use is of a combined agent, including a histone deacetylase inhibitor, such as phenylbutyrate, and a angiotensin converting enzyme inhibitor, such as captopril or pharmaceutically acceptable salts thereof.

As recognized by those skilled in the art, the effective dose would vary depending on route of administration, excipient usage, and the possibility of co-use of phenylbutyrate with other therapeutic treatments, such as the use of angiotensin converting enzyme inhibitors other than captopril, or angiotensin converting enzyme receptor blockers, or beta adrenergic receptor blockers, or diuretics, or other standard heart failure drug treatments. Effective amounts and treatment regimens for any particular subject (e.g., human, dog, horse or cat) will also depend upon a variety of other factors, including the activity of the specific compound employed, age, body weight, general health status, sex diet time of administration, rate of excretion, severity and course of the disease, and the patient's disposition to the disease, but are usually from 1.0 to 30 grams of phenylbutyrate per day irrespective of the manner of administration.

In order that the invention described herein may be more readily understood, the following examples are set forth. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting this invention in any manner.

Example 1 A Combined Histone Deacetylase Inhibitor and Angiotensin Converting Enzyme Inhibitor Treatment is Effective for Heart Failure

Adult male spontaneously hypertensive rats (SHR) rats were purchased as retired breeders from Taconic Inc and boarded in the animal facility at the Boston VA Healthcare system until the time of study (18 to 24 months of age). Each rat was caged alone and allowed free access to chow and water. Animals were monitored several times per week for evidence of tachypnea and labored respiration; when these clinical findings were clearly in evidence, animals were either killed and studied or treated with captopril and/or phenylbutyrate. Groups of animals were treated by adding the angiotensin converting enzyme inhibitor captopril to the drinking water (2 g/L to drinking water) and/or phenylbutyrate to the drinking water (6 g/L to the drinking water; sodium 4-phenylbutyrate (Triple Crown America, Inc.)) when clinical evidence of impaired cardiac function was detected. Treatment was continued for 30 days, the animals closely monitored and then studied. A control group of age-matched, untreated SHR rats was used for comparison. At autopsy, animals were examined for pleural or pericardial effusions, atrial thrombi, and right and left ventricular hypertrophy. The heart was lightly blotted and weight, and individual cardiac chambers were quickly dissected, and chamber weight recorded. Left ventricular tissue samples were rapidly frozen and stored in liquid nitrogen for later mRNA analysis. Subsequently, total RNA was isolated from all left ventricular samples and subjected to individual Affymetrix (230 2.0) array analysis (performed at the Genomics Laboratory of Boston University School of Medicine, Boston Mass.). Six individual animals were included in each experimental group.

mRNA Analysis of Left Ventricular Tissue from SHR with Heart Failure Treated with Phenylbutyrate and Captopril or Without Treatment

Statistical analysis identified 1431 genes from the 28,000 probesets surveyed that were significantly different (p<0.05) between non-failing SHR and SHR with heart failure. Of these, 713 genes were up-regulated and 718 were down-regulated. FIGS. 7A-7B provide a visual summary of the quantitative changes in transcripts observed with heart failure (SHR-F) compared to non-failing gene expression (SHR-F vs. SHR-NF) and those effects of treatment (SHR-F+C and SHR-F+CP) relative to un-treated SHR with heart failure. Fewer than 2% of transcripts were significantly modified with captopril treatment (p<0.01). Captopril treatment (alone) reversed expression of 27 identified genes 12 were down-regulated and 15 were up-regulated which were overexpressed and repressed, respectively, with heart failure. FIGS. 8A-8B provide a summary of the number of identified transcripts whose expression was reversed following treatment relative to un-treated SHR with failure. In contrast, phenylbutyrate when combined with captopril treatment reversed expression of more than 47% of heart failure-induced changes in gene expression (p<0.01). The expression of 665 genes were reversed when phenylbutyrate was added to captopril therapy (i.e. 308 were down-regulated and 357 up-regulated that were induced and repressed, respectively, with failure; see FIGS. 8A-8B). Cardiac left ventricular tissue showed a dramatic change in global gene expression, including induction or repression of numerous genes affecting multiple cellular processes, as presented in FIGS. 5A and 5B, which was strongly linked to the improved physiologic state. Analysis of pooled mRNA samples of right ventricular tissue indicate relatively few genes were differentially expressed between LV and RV tissue either with failure or treatment (data not shown). LV and RV are similarly affected by heart failure and amenable to therapeutic treatment effects. Taken together, these findings are consistent with the concept that phenylbutyrate acts as a wide scale inhibitor of transcription to reverse aspects of adverse remodeling and dysfunction associated with heart failure.

Example 2 The Ventricular Mass Increases with Heart Failure

There is an increase in left and, to a greater extent, right ventricular weight with heart failure in SHR rats. See Table 1 and FIGS. 4A and 4B.

Table 1 (below) summarizes body weight, cardiac chamber weight, and ratios with and without treatment. Referring to Table 1, four groups of six SHR/group were examined: non-treated SHR with heart failure (SHR-F), age matched controls without heart failure (SHR-NF), and SHR with heart failure treated with the angiotensin converting enzyme inhibitor captopril (2 g/L in drinking water) (SHR-F+C) or captopril combined with the histone deacetylase inhibitor phenylbutyrate (6 g/L in the drinking water) for 30 days (SHR-F+CP). The results show changes in the pathological tissue weight between the SHR with heart failure (SHR-F) and non-failing SHR groups (SHR-NF), and the group treated with phenylbutyrate, and, to a far lesser extent, the captopril treated group. It was observed that SHR rats with heart failure had enlarged hearts and increased cardiac chamber weights relative to non-failing SHR group and the captopril treated SHR groups. In contrast, the rats treated with phenylbutyrate and captopril had far smaller cardiac chamber weights. There were no significant differences in body weight among the experimental groups. Indices of cardiac hypertrophy (i.e. LV/body weight ratio and RV/body weight ratios) were reduced with phenylbutyrate treatment. Cardiac chamber weight to tibia length data confirmed these findings. See the following Table 1.

TABLE 1 BW LV wt. RV wt. LV/body wt. RV/body wt. (g) (mg) (mg) (ratio) (ratio) SHR-NF 404 ± 34 1.3 ± 0.1 0.23 ± 0.04⁺ 3.2 ± 0.3⁺ 0.57 ± 0.10⁺ SHR-F 362 ± 30 1.4 ± 0.1  0.41 ± 0.05*  3.9 ± 0.3*  1.12 ± 0.17* SHR-F + C 404 ± 28 1.3 ± 0.1  0.34 ± 0.07*⁺ 3.3 ± 0.3⁺  0.86 ± 0.20⁺* SHR-F + CP 403 ± 24   1.0 ± 0.1*^(+#) 0.25 ± 0.06⁺   2.5 ± 0.2*^(+#) 0.62 ± 0.12⁺ BW = body weight; HT wt. = total heart weight; LV wt. = left ventricular weight; RV wt. = right ventricular weight; LV/body wt. = left ventricular to body weight ratio; RV/body wt. = ratio of right ventricular to body weight ratio. Values are means ± SD. *p < 0.05 vs. SHR-NF; ⁺p < 0.01 vs. SHR-F; ^(#)p < 0.01 vs. SHR-F + C

Phenylbutyrate as an adjunct treatment for heart failure mediates regression of adverse cardiac remodeling.

Example 3 Effect of Phenylbutyrate and Captopril Treatment on Myocardial Muscle Function of SHR with Heart Failure

At the time of sacrifice, left ventricular papillary muscles were dissected free and mounted in an isolated muscle bath containing oxygenated physiologic solution for assessment of mechanical function. Muscle mechanics were measured under computer control of force or length of the preparation. Force and length data were sampled at a rate of 1 kHz and stored on disk for later analysis.

Table 2 (below) summarizes the changes in left ventricular papillary muscle mechanical function obtained from SHR with heart failure (SHR-F), age-matched non-failing SHR (SHR-NF), and SHR with heart failure treated at the time of failure with captopril (SHR-F+C; 2 g/l in the drinking water) or captopril and phenylbutyrate (SHR-F+CP; 6 g/l in the drinking water). Treatment was continued for 30 days and then the animals studied.

TABLE 2 AT +dT/dt RT_(1/2) V_(1.0) Kwm (g/mm²) (g/mm²/s) (ms) (muscle length/s) (stiffness) SHR-NF 4.3 ± 0.3  43.1 ± 8.6 188 ± 14  1.4 ± 0.4  53.2 ± 4.3  SHR-F 2.5 ± 0.7* 22.2 ± 6.8 144 ± 14* 0.7 ± 0.2*  73.5 ± 14.1* SHR-F + C 2.7 ± 0.4* 28.4 ± 8.0 148 ± 25* 0.9 ± 0.2* 73.9 ± 4.2* SHR-F + CP  5.3 ± 1.8^(+#)  56.3 ± 9.0^(+#) 184 ± 21   1.6 ± 0.2^(+#) 64.6 ± 10.6 AT = active tension; +dT/dt = rate of tension development; RT_(1/2) = relaxation time index; V_(1.0) = shortening velocity; Kwm = whole muscle stiffness. Values are means ± SD. *p < 0.05 vs. SHR-NF; ⁺p < 0.01 vs. SHR-F; ^(#)p < 0.01 vs SHR-F + C

Captopril treatment of SHR with heart failure did not restore contractile function or reduce passive muscle stiffness. In contrast, when phenylbutyrate was added to captopril treatment contractile dysfunction was reversed.

In conclusion, phenylbutyrate is a active compound for the treatment of heart failure when used in conjunction with captopril. Combined compound treatment ameliorates changes in cardiac gene expression, chronic enlargement of ventricular mass and depression of cardiac contractile muscle function associated with heart failure. The present invention also relates to a method for the treatment of humans or animals afflicted with either acute or chronic heart failure, comprising administering to the subject an effective amount of a histone deacetylases inhibitor combined with a angiotensin converting enzyme inhibitor in particular phenylbutyrate and captopril, respectively, or pharmaceutically acceptable salts thereof and optionally a suitable excipent.

All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. For example, compounds structurally and functionally analogous to phenylbutyrate described above can also be used in combination with other angiotensin converting enzyme inhibitor other than captopril to practice the present invention. Thus, other embodiments are also within the claims.

DISCUSSION

We have shown (Reference 7) that treatment of spontaneously hypertensive rats with heart failure of a combined mixture phenylbutyrate and captopril together administered in the drinking water for 30 days, but not captopril alone, can reverse expression of 655 heart failure associated genes (determined by Affymetrix gene chip array analysis and results confirmed by real time PCR of selected transcripts) which was associated with reduced right and left ventricular (LV) hypertrophy (see FIGS. 4A and 4B), improved LV ejection fraction in vivo (determined by echocardiogram; see FIGS. 1A-1B and 2A-2B), LV papillary muscle function (see FIGS. 6A and 6B) and beta-adrenergic responsiveness in vitro (data not shown). This study suggests that reestablishing gene transcription through phenylbutyrate's inhibition of histone deacetylase (HDAC) and DNA methylation represents a promising adjunct treatment when combined with ACE inhibition that can reverse many of the adverse pathophysiologic changes found in the failing heart. The findings that phenylbutyrate improves gene transcription and clinical symptoms in a rat model of heart failure, indicates that this combined treatment strategy may be considered for therapeutic targeting of cardiac gene transcription for the treatment of patients with heart failure. It is likely that epigenetic mechanisms may underlie many pathophysiological changes with heart failure. The histone deacetylase inhibitory actions of phenylbutyrate appears to be an effective adjunctive treatment to modulate transcription and cause changes in expression of many heart failure associated genes. FIG. 9 describes the proposed mechanism by which phenylbutyrate is likely to modulate cardiac gene transcription. Phenylbutyrate by inhibiting histone deacetylase (HDAC) promotes gene transcription and thereby improves cardiac function (see FIG. 10).

Treatment Reverses Intrinsic Myocardial Dysfunction and Genotypic Changes Associated with Heart Failure

Current treatment for heart failure stabilizes hemodynamics but improvement in intrinsic cardiac muscle function has not been demonstrated. We studied isolated papillary muscle function in the spontaneously hypertensive rat (SHR) which develops heart failure (HF) between 18-24 month of age. Four groups of SHR each consisting of six animals were examined: non-treated SHR with HF (SHR-F), age matched controls without HF (SHR-NF), and SHR with HF that were treated with the ACE inhibitor captopril (2 g/L in drinking water) (SHR-F+C) or captopril combined with the histone deacetylase inhibitor phenylbutyrate (6 g/L in drinking water) for 30 days (SHR-F+CP). FIG. 8 shows the primary pharmacological drug actions of captopril and phenylbutyrate. Following treatment, hearts were isolated and contractile function of LV papillary muscles studied. LV weight and histology were assessed. RNA was isolated from 24 LV samples and subjected to individual Affymetrix (230 2.0) array analysis. Table 3 (below) summarizes the physiological, histological and gene expression findings from this study. Quantitative histological examination of myocardial fibrosis revealed no differences in fibrosis among treatment groups. Statistical analysis identified 1431 genes from the 28,000 genes surveyed that were significantly different (p<0.05) between SHR-F and SHR-NF: 713 genes were upregulated and 718 were downregulated in SHR-F as compared to SHR-NF (see FIGS. 7A-7B). Captopril was found to significantly reverse the change in expression of 141 genes (9.8%) while phenylbutyrate+captopril reversed 1131 (79.0%) (both p<0.01 vs. SHR-F; see FIGS. 7A-7B). Table 4 lists the eight identified genes whose expression was significantly altered with heart failure and subsequently reversed following captopril treatment. In contrast to captopril treatment alone, Table 5a and 5b lists the many identified gene changes that were up-regulated and down-regulated, respectively, with combined phenylbutyrate and captopril treatment. Although combined treatment induced changes in gene expression involving many cell processes, many changes were closely associated with suppression of death associated processes (i.e apoptosis and proteolysis related gene expression). Thus, captopril stabilizes hemodynamics with heart failure and provides a window to treat underlying cardiac dysfunction, but did not improve intrinsic contractile function. The addition of phenylbutyrate to ACE inhibitor resulted in reversal of changes in transcripts, hypertrophy and myocardial dysfunction associated with heart failure.

TABLE 3 LV/Body V_(0.1) Gene Weight AT (muscle Expression (ratio) (g/mm²) lengths/s) Fibrosis (%) (number) SHR-NF 3.6 ± 0.4 4.3 ± 0.3  1.4 ± 0.4  12.5 ± 3.5  SHR-F 3.8 ± 0.3 2.5 ± 0.7* 0.7 ± 0.2* 21.9 ± 3.6* 713↑* 718↓* SHR-F + C 3.3 ± 0.4 2.7 ± 0.4* 0.9 ± 0.2* 22.8 ± 3.1* 42↓^(†) 99↑^(†) SHR-F + CP  2.7 ± 0.2*^(,†)  5.3 ± 1.8^(†,) ^(§)  1.6 ± 0.2^(†,) ^(§) 18.4 ± 4.0* 485↓^(†,§) 646↑^(†,§) AT = active tension; V_(0.1) = shortening velocity mean ± SD. *p < 0.05 vs. SHR-NF. ^(†)p < 0.01 vs. SHR-F. ^(§)p < 0.01 vs. SHR-F + C.

TABLE 4 List of Identified Gene Changes with Heart Failure, Reversed by Captopril Treatment (p < 0.01), Ranked by Fold Change. Fold change Func- Identifier Gene Description F/NF tion NM_017272 aldehyde dehydrogenase family 1, 2.34 B NM_031145 calcium and integrin binding 1 1.27 CA NM_138823 protein phosphatase 1, regulatory 1.24 CS (inhibitor) subunit 2 BG663093 sequestosome 1 1.23 TT NM_017038 protein phosphatase 1A, 1.23 CS magnesium dependent, alpha isoform NM_017039 protein phosphatase 2a, 1.11 CS catalytic subunit, alpha isoform BI282044 acetyl-CoA transporter 0.77 EM NM_012555 v-ets erythroblastosis virus E26 0.67 TT oncogene homolog 1

-   -   Expression is given as a fold difference of expression present         in the SHR-F transcriptional profile relative to that of SHR-NF.         B, Biosynthesis, CA, cell adhesion or binding activity; CS, cell         signaling/communication; EM, energy metabolism; TT,         transcription/translation regulatory activity.

TABLE 5a Transcripts Up-Regulated with Heart Failure (p < 0.05) and Significantly Down-Regulated with Combined Treatment (p < 0.01). Up-Regulated Transcripts Ranked by Fold Change Fold change Func- Identifier Description F/NF tion Up-Regulated with failure < with Rx BF289368 lipopolysaccharide binding protein 6.11 CD NM_053611 nuclear protein 1 3.11 TT NM_053326 enigma homolog 2.96 CS NM_031832 lectin, galactose binding, soluble 3 2.9 CA BF419200 CCAAT/enhancer binding protein 2.73 TT (C/EBP), delta NM_012620 serine (or cysteine) proteinase 2.77 CS inhibitor, clade E, member 1 AF245040 Dickkopf homolog 3 2.68 CS NM_017080 hydroxysteroid 11-beta dehydrogenase 1 2.65 B D63648 phospholipase B 2.55 EM AI716896 secreted frizzled-related protein 1 2.54 D NM_017320 cathepsin S 2.50 D BI304009 lysyl oxidase 2.42 PM NM_017272 aldehyde dehydrogenase family 1, 2.34 B SubfamilyA4 NM_021663 nucleobindin 2 2.23 CA BI298314 von Willebrand factor 2.20 CD NM_020104 fast myosin alkali light chain 2.19 S NM_134401 cartilage acidic protein 1 2.19 CA NM_012905 aortic preferentially expressed gene 1 2.19 TT AF290212 calcium channel, voltage- 2.11 T dependent, T type, alpha 1G subunit NM_017237 ubiquitin carboxy-terminal hydrolase L1 2.07 B NM_053629 follistatin-like 3 1.97 CS AB032395 decay accelarating factor 1 1.90 CD AB049572 sphingosine kinase 1 1.8 D NM_012846 fibroblast growth factor 1 1.84 G AJ277077 damage-specific DNA binding protein 1 1.84 TT NM_031514 Janus kinase 2 1.82 CS NM_031684 solute carrier family 29 (nucleosid 1.79 T transporters), member 1 AW253722 RAB13, member RAS oncogene family 1.73 CS BI296610 glutamine synthetase 1 1.68 B NM_031740 UDP-Gal:betaGlcNAc beta 1.68 B 1,4-galactosyltransferase, polypeptide 6 NM_053555 vesicle-associated membrane protein 5 1.67 S BI297004 PX domain containing serine/threonine 1.64 CS L38615 glutathione synthetase 1.64 B AF051335 reticulon 4 1.59 CD BI284461 v-maf musculoaponeurotic 1.59 TT fibrosarcoma oncogene family, protein K AI176519 immediate early response 3 1.58 G D88666 phosphatidylserine-specific 1.58 M phospholipase A1 BI303655 ATPase, Na+/K+ transporting, 1.56 T beta 3 polypeptide NM_053857 eukaryotic translation initiation 1.53 TT factor 4E binding protein 1 J02612 UDP glycosyltransferase 1 family, 1.52 B polypeptide A1 /// UDP * (Ugt1a6 /// Ugt1a7 /// Ugt1a4 /// Ugt1a8 /// Ugt1a2 /// Ugt1a1) glycosyltransferase 1 family, polypeptide A6 /// UDP glycosyltransferase 1 family, polypeptide A7 /// UDP glycosyltransferase 1 family, polypeptide A8 /// UDP glycosyltransferase 1 family polypeptide A2 /// UDP glycosyltransferase 1 family polypeptide A4 /// UDP glycosyltransferase 1 family polypeptide A11 /// UDP glycosyltransferase 1 family, polypeptide A5 NM_012636 parathyroid hormone-like peptide 1.51 CS NM_017334 cAMP responsive element modulator 1.50 TT AI176595 cathepsin L 1.50 D AI576297 programmed cell death 4 1.49 D NM_031576 P450 (cytochrome) oxidoreductase 1.47 EM BI278802 prion protein 1.46 CD NM_017022 integrin beta 1 1.45 CA U75920 microtubule-associated protein 1b 1.43 S NM_053653 vascular endothelial growth factor C 1.42 G L12407 dopamine beta hydroxylase 1.41 B BI303379 tumor necrosis factor receptor 1.41 CD superfamily, member 12a BM391807 acyl-Coenzyme A dehydrogenase 1.38 EM family, member 9 NM_031646 receptor (calcitonin) activity 1.38 CS modifying protein 2 BE112590 discoidin domain receptor family, 1.37 CS member 1 NM_134449 protein kinase C, delta binding protein 1.31 CG BE097791 meningioma expressed antigen 5 1.31 B (hyaluronidase) NM_031823 Wolfram syndrome 1 1.30 B BM383722 NCK-associated protein 1 1.29 D NM_012734 hexokinase 1 1.28 EM NM_019124 rabaptin 5 1.28 D NM_031145 calcium and integrin binding 1 1.27 CA AW527957 Bcl2-like 2 1.25 D BG671677 amyloid beta (A4) precursor protein. 1.25 CD AI008441 phosphogluconate dehydrogenase 1.25 EM NM_138823 protein phosphatase 1, regulatory 1.24 CS (inhibitory) subunit 2 BG663093 sequestosome 1 1.23 TT AI237597 heat shock protein 1, alpha 1.23 PM BI282866 lamin A 1.23 S NM_017038 protein phosphatase 1A, 1.23 CS magnesium dependent, alpha isoform NM_017039 protein phosphatase 2a, 1.22 TT catalytic subunit, beta isoform M83143 sialyltransferase 1 1.22 PM BG668463 nitrilase 1 1.21 B NM_053739 beclin 1 (coiled-coil, myosin-like 1.20 CD BCL2-interacting protein) NM_022531 desmin 1.18 S NM_053522 ras homolog gene family, member Q 1.18 CS M86443 hypoxanthine guanine phosphoribosyl 1.17 B transferase BF283010 syntaxin 12 1.17 T NM_053290 phosphoglycerate mutase 1 1.16 EM NM_031785 ATPase, H+ transporting, 1.16 T lysosomal (vacuolar proton pump), subunit 1 BE329377 Jun D proto-oncogene 1.15 TT AA850867 gamma sarcoglycan 1.13 S M62763 sterol carrier protein 2 1.2 B NM_017039 protein phosphatase 2a, catalytic 1.11 TT subunit, alpha isoform NM_017039 protein phosphatase 2a, catalytic 1.11 TT subunit, alpha isoform NM_022523 CD151 antigen 1.07 CA CA, cell adhesion or binding activity; CD, cell defense (i.e. immunity/oxidative stress); CS, cell signaling/communication; D, death associated; E, enzyme activity; G, cell growth/maintenance; M metabolism; PM, protein or structural modifier; S, cell structure (i.e. ECM) or function (i.e. contraction); T, transport; TT, transcription/translation regulatory activity.

TABLE 5b Transcripts Down-Regulated with Heart Failure (p < 0.05) and Significantly Up-Regulated with Combined Treatment (p < 0.01). Down-Regulated Transcripts Ranked by Fold Change Fold change Func- Identifier Description F/NF tion Down-Regulated with failure > with Rx AI535411 myosin, heavy polypeptide 7, 0.18 S cardiac muscle, beta AI103845 UDP-N-acetyl-alpha-D- 0.28 PM galactosamine: polypeptide N- acetylgalactosaminyltransferase 13 AF385402 potassium channel, subfamily K, 0.33 T member 2 M74449 potassium voltage-gated channel, 0.34 T shaker related subfamily, member 2 AF134409 RASD family, member 2 0.35 CS NM_031730 potassium voltage gated channel, 0.37 T Shai-related family, member 2 J02997 dipeptidylpeptidase 4 0.42 D NM_022604 endothelial cell-specific molecule 1 0.46 G NM_019174 carbonic anhydrase 4 0.48 B BE098261 solute carrier family 25 0.50 T (mitochondrial oxodicarboxylate carrier) NM_033352 ATP-binding cassette, sub-family 0.51 EM D(ALD) member 2 NM_017239 myosin, heavy polypeptide 6 0.52 S AA799574 L-3-hydroxyacyl-Coenzyme A 0.52 EM dehydrogenase, short chain X95189 acyl-CoenzymeA oxidase 2, 0.52 EM branched chain AI411446 pyruvate dehydrogenase 0.53 EM phosphatase isoenzyme 2 NM_012506 ATPase Na+/K+ transporting, 0.53 EM alpha 3 polypeptide BE113154 FK506 binding protein 4 0.56 PM AA79 421 protein kinase C, epsilon 0.58 CS BE113289 peroxisome proliferator-activated 0.59 TT receptor gamma coactivator 1 beta NM_031715 phosphofructokinase, muscle 0.66 EM BG378763 glycerol-3-phosphate dehydrogenase 2 0.65 EM NM_017328 phosphoglycerate mutase 2 0.61 EM AI411979 carnitine acetyltransferase 0.61 EM NM_012930 carnitine palmitoyltransferase 2 0.67 EM D88891 brain acyl-CoA hydrolase 0.63 EM BI276974 valyl- tRNA synthetase 2-like 0.64 TT BE111733 hormone-regulated proliferation- 0.65 D associated 20 kDa protein NM_133606 enoyl-Coenzyme A hydratase/3- 0.65 EM hydroxyacyl Coenzyme A dehydrogenase NM_022936 epoxide hydrolase 2, cytoplasmic 0.65 B NM_019156 vitronectin 0.66 CD BF555973 triadin 0.66 S NM_022400 branched chain aminotransferase 2, 0.67 EM mitochondrial AA945624 NAD(P)H dehydrogenase, quinone 2 0.67 EM NM_012555 v-ets erythroblastosis virus E26 0.68 TT oncogene homolog 1 NM_012793 guanidinoacetate methyltransferase 0.68 EM AA900057 PDZ protein Mrt 1 0.69 CS BE113377 potassium inwardly-rectifying 0.70 TT channel, subfamily J, member 11 AI411530 aminoacylase 1 0.70 D AI235510 B-cell receptor-associated protein 37 0.70 TT M29853 cytochrome P450, family 4, subfamily b 0.74 EM BM389548 guanine nucleotide binding protein 0.74 CS (G protein), gamma 5 subunit AI177031 ER transmembrane protein Dri 42 0.75 S NM_031326 transcription factor A, mitochondrial 0.77 TT BI282044 acetyl-CoA transporter 0.78 EM NM_053722 CLIP associating protein 2 0.78 TT BI296061 nuclear transcription factor Y-gamma 0.78 TT BI289129 tyrosine kinase receptor 1 0.78 S NM_053483 karyopherin (importin) alpha 2 0.78 T (Tpm1) tropomyosin 1, alpha 0.79 S U91449 potassium inwardly-rectifying 0.81 T channel, subfamily J, member 3 AA892297 histone deacetylase 2 0.83 TT NM_031616 zinc finger 265 0.88 TT NM_012904 Annexin A1 0.89 EM AA859652 solute carrier family 16, 0.90 T (monocarboxylic acid transport)membrane 7 NM_022385 ADP-ribosylation factor- 1 0.92 T CA, cell adhesion or binding activity; CD, cell defense (i.e. immunity/oxidative stress); CS, cell signaling/communication; D, death associated;, E, enzyme activity; G, cell growth/maintenance; M metabolism; PM, protein or structural modifier; S, cell structure (i.e. ECM) or function (i.e. contraction); T, transport; TT, transcription/translation regulatory activity.

The preliminary findings which have been presented in abstract (Reference 7) form and soon to be submitted for scientific publication describe methods designed to administer a compound mixture including sodium phenylbutyrate and captopril dissolved in the drinking water of animals in need of treatment for heart failure. However, solid dosage forms for oral administration including capsules, tablets, pills, touches, lozenges, powders and granules, containing the active ingredients or intravenous administration may be required to provide maximum therapeutic effectiveness to humans in need of treatment for heart failure. Furthermore, sodium salts are contraindicated in patients with heart failure, it is likely therefore that a compound of these active ingredients including an ester, rather than the sodium salt may be more clinically efficacious.

The description in this application is specifically directed to phenylbutyrate in combination with an angiotensin converting enzyme inhibitor, such as captopril, in the treatment of heart failure and cardiac hypertrophy, as non-limiting examples and is not intended to limit the scope of the invention.

While this invention has been described as having preferred sequences, ranges, ratios, steps, order of steps, materials, structures, symbols, indicia, graphics, color scheme(s), shapes, configurations, features, components, or designs, it is understood that it is capable of further modifications, uses and/or adaptations of the invention following in general the principle of the invention, and including such departures from the present disclosure as those come within the known or customary practice in the art to which the invention pertains, and as may be applied to the central features hereinbefore set forth, and fall within the scope of the invention and of the limits of the claims appended hereto or presented later. The invention, therefore, is not limited to the preferred embodiment(s) shown/described herein.

REFERENCES

The following references, and those cited in the disclosure herein, are hereby incorporated herein in their entirety by reference.

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What is claimed is:
 1. A method of preventing cardiac gene expression changes associated with heart failure, comprising: administering to a subject in need thereof a therapeutically effective amount of a composition including a histone deacetylase (HDAC) inhibitor or a pharmaceutically acceptable salt or ester thereof, and an angiotensin converting enzyme (ACE) inhibitor, angiotensin receptor blocker, beta adrenergic receptor blocker, or a pharmaceutically acceptable salt or ester thereof.
 2. A method of preventing myocardial dysfunction, comprising: administering to a subject in need thereof a therapeutically effective amount of a composition including a histone deacetylase (HDAC) inhibitor or a pharmaceutically acceptable salt or ester thereof, and an angiotensin converting enzyme (ACE) inhibitor, angiotensin receptor blocker, beta adrenergic receptor blocker, or a pharmaceutically acceptable salt or ester thereof. 